Plants have two respiratory electron transport chains from ubiquinone to oxygen: the cytochrome c pathway and the alternative pathway. The former is common to all eukaryotes, while the latter is found in plants and some lower eukaryotes, but not mammals. A distinct feature of the alternative pathway is that only one protein, alternative oxidase (AOX), is involved in transport of electrons from ubiquinone directly to oxygen. The alternative pathway does not generate a transmembrane potential in contrast to the cytochrome c pathway. Because of this seemingly "wasteful" property, the biological function of the alternative pathway has long been an enigma. Although, the cDNAs for AOX have been cloned from a number of plants, little is known about its function. To further study the biological function of AOX, transgenic potato plants constitutively overexpressing the enzyme were created in our laboratory. Their phenotype resembled that of the wild type plants, although there was about a two-fold increase in the capacity of the alternative pathway. However, a new AOX band could be seen on the Western blots of total mitochondrial protein. The band migrated about 1kD slower than the doublet of bands representing AOX in leaves of the normal plants and was present in much higher concentrations. One of the explanations for this phenomenon could be that the upper band represents a precursor for the faster migrating AOX doublet. The overexpressing plants may accumulate the precursor due to saturation of a putative processing/modifying system by a vast excess of AOX protein. The precursor could be processed at the N-terminus, C-terminus and/or modified to produce the faster migrating AOX bands. We are currently using MALDI-MS and peptide mass mapping to investigate the structure of this isoform of AOX.